This proposal has three objectives: (a) to study the time course of formation and intracellular movement of endosomes in cultured ovarian granulosa cells containing fluorescent derivatives of human chorionic gonadotropin (hCG) by time lapse video image intensification microscopy (TLVIM); (b) to determine the mode of uptake and fate of human chorionic gonadotropin by immunoelectron microscopy; and (c) to evaluate the role of endosome acidification and cytoplasmic microtubules in the intracellular movement of endosomes. A better understanding of the mechanisms employed by cells to bind, internalize, and process physiologically relevant hormones should aid in the interpretation of disease processes based upon defects in these activities as well as suggest new avenues for the effective delivery of therapeutic agents to target cells to manage certain disease states. These studies are based on the observations that ligand-bearing endosomes are rapidly acidified and undergo directed movements within the cytoplasm that may function in the delivery of ligands to lysosomes. Fluorescent derivatives of hCG will be characterized with respect to their binding efficiency and biological activity. The behavior of these probes in living cultured ovarian granulosa cells will be monitored by TLVIM with regard to the characteristics of endosome movement and the relationship of hCG-bearing endosomes with lysosomes. hCG distribution at the cell surface and intracellularly will be determined using immunoelectron microscopic localization on primary cultures of hCG treated granulosa cells. Finally, fluorescence microscopy will be used in conjunction with pharmacological inhibitors of endosome acidification or microtubule function to probe the possible importance of these components in the normal catabolism of hCG.